This application is a 371 of PCT/SE98/00387 filed Mar. 4, 1998.
The present invention relates to matrices which can be used for separation of one or more components in a mixture of components. There are also other uses of the particular type of matrices. The separation means that a liquid containing the component(s) is contacted with the particular type of matrix, wherein the component(s) to be removed is (are) partitioned to the matrix and thereby separated from the remaining components which are differently partitioned to the matrix. By the expression xe2x80x9cpartitioned to the matrixxe2x80x9d is meant that components bind or otherwise are adsorbed on (in) the matrix.
The separation methodology may be in form of chromatography on a monolithic matrix or on a packed or fluidized bed of particles or as a batchwise process with suspended particles. Partition to the matrix may be based on affinity or molecular size/molecular shape, such as in affinity and gelchromatography, respectively.
By separation is also meant desalting, buffer exchange, concentration and the like, wherein a separation matrix is contacted with a liquid containing something to be removed.
In the liquid containing the components to be separated, a convective mass transport occurs because of streaming or turbulence. In relation to this mass transport, matrices used for separation according to the above typically show two kind of environments: 1) an environment in which convective mass transport occurs (convective environment) and 2) an environment in which only diffusive transport occurs (diffusion environment). The two environments usually are in contact with each other via the liquid used through openings preventing convective mass transport.
Monolithic matrices and particulate matrices may show both types of environments. For monolithic matrices in the form of through flow pores and diffusion pores, respectively. A further convective environment for particles is the external environment around the particles, i.e. the void volume between the particles in the packed beds and surrounding liquid in suspensions and fluidized beds. Matrices intended for chromatography and showing both through flow and diffusion pores have probably been utilized for a long time without ability to optimally use the pore types. See, for example, WO-A-9100762 (Perseptive Biosystems Inc.). Matrices based on polysaccharides and showing both types of pores have been described in WO-A-9319115 (Pharmacia Biotech AB).
The size limit for the pores to function as through flow or diffusion pores is a consequence of the mobility an viscosity of the liquid (for example flow rate), characteristics of the pore surface, possible coating on the surface restricting the flow giving convective mass transport etc. Sizes of through flow pores which are of interest for separation purposes are within the interval 0.4-1,000 xcexcm. The size of diffusive pores is a consequence of the same variables as those of the through flow pores. For separation purposes the diffusive pores should typically be less than 1 xcexcm. The analogous holds also for the openings between the diffusion environment and the convective environment.
Technical Background: Advantages With the Invention and Problems Solved by the Invention.
In order to secure that a pure product is obtained, several different complementary chromatographic techniques are often used serially, e.g. gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, bioaffinity chromatography. Several sequential chromatographic steps are cumbersome and costly, and especially in an industrial scale the costs may limit the ability to accomplish purification.
Many attempts have been made to create multifunctional chromatographic media in order to decrease the number of purification steps, inter alia, by providing the chromatographic materials with different functional groups giving different types of physical/chemical interactions with the substances to be purified. In practice, different interactions often counteract each other leading to an impaired result (for example a combination of electrostatic and hydrophobic interaction).
Ion exchange on particulate ion exchangers often leads to aggregation of the matrix particles in the presence of macromolecules of opposite charge compared to the particles. These problems are due to surface charges and are accentuated in batchwise suspension procedures and fluidized beds. In this context, there is especially mentioned cell culture supernatants and other sample solutions containing whole cells and/or parts thereof, including microorganisms of different types.
In the present invention, it has been possible to achieve co-operation of different separation principles on the same chromatographic medium and in this way reduce the number of necessary separation steps in a purification process.
Known Matrices
Porous particles of cross-linked agarose coated with dextran are marketed by Amersham Pharmacia Biotech AB (Uppsala, Sweden) under the designation Superdex(copyright). In the production of Superdex(copyright), dextran is used having a molecular weight distribution which allows the dextran to be present in interior as well as in exterior parts of the particles.
Pore surfaces in membranes/matrices can be coated with polymers of such molecular weight that clogging of the pores is prevented (EP-A-221046, Monsanto).
Multifunctional matrices of a different construction than the invention have been described previously. See U.S. Pat. No. 454,485 (Purdue University; Hagestam and Pinkerton), U.S. Pat. No. 5,522,994 (Cornell Res. Found.; and Svec), WO-A-9409063 (Cornell Res. Found.; Frechet and Svec), WO-A-9408686 (Cornell Res. Found.; Frechet, Smigol and Svec) och WO-A-9317055 (Cornell Res. Found.; Frechet and Hosoya).
The objects of the invention are to provide:
1. Gel filtration media having improved selectivity with regard to the discrimination between components having different size and shape.
2. Porous particles or porous monolithic structures whose inner environment differs from their surface environment in regard to type of functional group, charge, hydrophobic/hydrophilic environment etc.
3. Simplified separation methods based on gel filtration, partition chromatography and affinity, including ion exchange, hydrophobic interaction, bioaffinity interactions/reactions etc., especially in regard to liquid chromatography.
4. New particles or monolithic structures with a surface layer inside or outside the matrix which are delimiting smaller inner pores (micropores) from larger pores (macropores) or from the surrounding of the matrix. This surface layer shall prevent compounds/components that are above a certain size from passing through and may also prevent larger compounds/components to attach to the matrix (surface layer, lock). The adsorption characteristics of the micropores may also be different from those of the surface layer so that components, penetrating into the micropores, do not adhere in/on the surface layer.